Buscar

DEWES & RIBBONS, 1964

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Você viu 3, do total de 24 páginas

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Você viu 6, do total de 24 páginas

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Você viu 9, do total de 24 páginas

Faça como milhares de estudantes: teste grátis o Passei Direto

Esse e outros conteúdos desbloqueados

16 milhões de materiais de várias disciplinas

Impressão de materiais

Agora você pode testar o

Passei Direto grátis

Prévia do material em texto

BACrERIOLOGICAL REVIEWS
Vol. 28, No. 2, pp. 128-149 June, 1964
Copyright @ 1964 by the American Society for Microbiology
Printed in U.S.A.
SOME ASPECTS OF THE ENDOGENOUS METABOLISM OF BACTERIA
E. A. DAWES AND D. W. RIBBONS
Department of Biochemistry, University of Hull, Hull, England
INTRODUCTION.................................................................................. 126
SUBSTRATES FOR ENDOGENOUS METABOLISM .................................................... 127
Role of PHB ................................................................................. 127
Biosynthesis of PHB ....................................................................... 130
Catabolism of PHB......................................................................... 131
Glycogenlike Reserves ......................................................................... 132
Amino Acid Pools............................................................................ 135
RNA Metabolism............................................................................. 136
Protein...................................................................................... 138
Other Potential Substrates..................................................................... 138
ENERGY OF MAINTENANCE...................................................................... 138
STARVATION AND SURVIVAL..................................................................... 142
Death........................................................................................ 143
Relationship Between Survival and Endogenous Substrates ...................................... 144
SUMMARY AND FUTURE OUTLOOK............................................................... 145
LITERATURE CITED............................................................................. 146
"One great use of a Review, indeed, is to
make men wise in ten pages, who have no
appetite for a hundred pages; to condense
nourishment, to work with pulp and essence,
and to guard the stomach from idle burden
and unmeaning bulk."
Sidney Smith
Edinburgh Review, 1825
INTRODUCTION
Current interest in the endogenous metab-
olism of microorganisms has been reflected by
two recent symposia (Focal Topic A7, Intern.
Congr. Microbiol., 7th, Montreal, 1962; Ann.
N.Y. Acad. Sci., vol. 102, art. 3, p. 515-793,
1963) and by our own previous survey (15). The
present article is intended to be a more discursive
review of the present situation in this area of
study and to deal in greater detail with those
aspects which were not previously elaborated.
Endogenous metabolism may be defined as
the total metabolic reactions that occur within
the living cell when it is held in the absence of
compounds or elements which may serve as
specific exogenous substrates. Some products of
endogenous metabolism may be released into
the surrounding medium and are often utilized
by the cells, sometimes resulting in regrowth, a
phenomenon which is discussed later. It is im-
portant to stress the viability and integrity of
the cell in this context, because some authors
have used the term "endogenous" loosely in
relation to the activities of cell extracts (see, for
example, 84).
It must also be recognized that the reactions
characteristic of endogenous metabolism may
continue in the presence of exogenous substrates,
and, since metabolism of the latter compounds
is usually effected after they have been taken
into the cell, in the final analysis the distinction
between the metabolism of endogenous and
exogenous substrates may become largely a
matter of semantics, although problems of com-
partmentation within the cell should be appre-
ciated (22a). This is particularly the case where
the oxidation of an exogenous substrate by a
washed suspension of microorganisms leads to the
assimilation of material which subsequently can
be utilized as an endogenous substrate. Analysis
of the status of the endogenous metabolism in
the presence of added substrates presents many
experimental problems, but these have already
been adequately reviewed (6, 15).
The possible significance of endogenous metab-
olism in relation to the survival of microor-
ganisms is a topic presently under study in
several laboratories. One view which may be
held is that endogenous metabolism occurs
simply because the organism cannot help it, and
it therefore bears no relationship, direct or other-
wise, to the period of survival (22). The other
extreme outlook is that the survival character-
126
ENDOGENOUS METABOLISM OF BACTERIA
istics are related directly to the endogenous
metabolic activities of the cell. The evidence
available at the time of writing suggests that,
for those organisms which have been studied,
the truth lies somewhere between these extremes,
as will be discussed later.
The existence of an energy of maintenance for
microorganisms has attracted considerable ex-
perimental attention of late. The idea that a
definite amount of energy must be expended to
enable a microbial cell to maintain its integrity
without growth or death occurring seems a
reasonable concept, and appears now to be sup-
ported by some evidence. The question of
whether a cell can exist in a condition such that
it is committed neither to growth nor to death is,
however, still a debatable issue. The experi-
mental problems posed by the death and regrowth
of bacterial cultures make difficult a conclusive
answer to this query at present.
Aspects of endogenous metabolism concerning
macromolecular turnover and cell differentiation
into spores were focal points of the symposium
published by the New York Academy of Sciences,
and will not be elaborated here. A symposium on
microbial reaction to environment has also
directed attention to the considerable variation
in composition and properties that can be pro-
duced by alteration of experimental conditions,
and additionally to the influence of the previous
history of the cells. Although the effect of some
environmental factors on endogenous metab-
olism has been investigated (65), there are many
other aspects of this same problem which have
not been explored. Indeed, there are so many
possible external influences on endogenous
metabolism that considerable caution should be
exercised in making generalized statements con-
cerning the endogenous metabolic behavior of
microorganisms.
The present review will pay particular atten-
tion to the role of poly-f3-hydroxybutyrate
(PHB) as a substrate for endogenous metab-
olism, because, as a storage compound unique to
bacteria, it holds a position of considerable
interest. Several groups of investigators have
demonstrated the presence of the polymer and
have studied its metabolism in various bacterial
species, but there has so far been no attempt to
correlate their findings in relation to endogenous
metabolism.
SUBSTRATES FOR ENDOGENOUS
METABOLISM
A consideration of possible substrates for
endogenous metabolism naturally focuses atten-
tion on energy-storage compounds. Wilkinson
(93) described three main classes of compounds
that could possibly act as energy-storage com-
pounds. These are polysaccharides, lipids (includ-
ing PHB), and polyphosphate; all occur in
widely varying amounts dependent upon the
particular species and the environmental condi-
tions. However, it is now apparent that there
are many other substrates for endogenous
metabolism; these include ribonucleic acid
(RNA) and protein (both are also subject to
turnover) and free amino acid and peptide pools.
Possible substrates not yet implicated in endoge-
nous metabolism include deoxyribonucleic acid
(DNA), cell-wall polymers, and cell-membrane
materials.
Role of PHB
The elucidation of the role of PHB in bacterial
endogenous metabolism is a fascinating story,
for, although the polymer was originally dis-
covered in 1927, a physiologicalrole for it was
not convincingly demonstrated until some 30
years later. PHB was first isolated by chloroform
extraction of an aerobic bacillus by Lemoigne
(42), following his earlier discovery that (3-
hydroxybutyrate was a metabolic product of the
organism. Since that time, PHB has been demon-
strated in a wide variety of bacterial species
(Table 1), and in some instances has been im-
plicated as an assimilatory product, from meas-
urements of gaseous exchange during photo-
metabolism of fatty acids and gross elemental
composition, by Gaffron (cited in 78). The quan-
tities of PHB within the bacterial cell vary enor-
mously; contents of up to 50% of the dry weight
have been recorded. It is a reserve that is peculiar
to microorganisms, and its functions, formation,
and synthesis have been studied extensively in
recent years, mainly by Doudoroff and Stanier,
Gibbons, Schlegel, Wilkinson, and their col-
laborators.
Sudanophilic granules present in bacteria were
considered by Lemoigne, Delaporte, and Croson
(43) to be composed of PHB; the data of Weibull
(88) subsequently supported this proposition.
However, it was not until the work of William-
VOL. 28, 1964 127
DAWES AND RIBBONS
son and Wilkinson (95) that the intracellular
lipid granules of Bacillus cereus and B. mega-
terium were demonstrated unequivocally to be
composed mainly of PHB (about 90%), although
neither this nor the remaining 10%, lipid is re-
sponsible for the sudanophilic properties of the
granules that are observed in situ. Macrae and
Wilkinson (46, 47) also studied the effect of
various cultural conditions on the synthesis of
PHB in B. megaterium. When the glucose con-
TABLE 1. Occurrence of poly-,3-hydroxybutyrate in
bacteria*
Species Reference
Bacillus megaterium............... 43, 77, 95
B. cereus.......................... 43, 95
B. mycoides....................... 43
B. anthracis....................... 43
Azotobacter chroococcum ........... 43
A. agilis.......................... 21
A. vinelandii...................... 21
Rhizobium sp...................... 21
Vibrio sp.......................... 31, 32
Chromobacterium violaceum........ 21
Pseudomonas solanacearum........ 32
P. antimycetica ................... 32
P. methanica...................... 39
P. pseudomallei................... 44
P. saccharophila .................. 19
Pseudomonas AMi ................ 59
Micrococcus halodenitrificans ...... 75, 76
Sphaerotilus natans ............... 58, 69
Hydrogenomonas sp................ 73
Rhodospirillum rubrum............ 19, 78
Rhodopseudomonas spheroides.... . 10
Chromatiurn okenii................ 72
Spirillum itersonnii............... 54
S. anulus......................... 54
S. serpens......................... 32, 54
* This is not a complete list of the bacterial
species that synthesize PHB.
centration in the growth medium was raised,
more of the polymer was synthesized; exhaustion
of the nitrogen source in the presence of excess
carbon and energy source permitted deposition
of about four times the amount of PHB as was
formed when glucose limited growth. Glucose,
pyruvate, and f-hydroxybutyrate were suitable
substrates for PHB production by washed sus-
pensions, but acetate, although itself unable to
effect synthesis (compare with Rhodospirillum
rubrum), enhanced PHB formation when supple-
menting other substrates. Anaerobic conditions
prevented PHB synthesis, as did dinitrophenol.
Concentrations of oxygen greater than 5% also
inhibited the assimilatory process. B. cereus, but
not B. megaterium, could effect PHB synthesis
under hydrogen, although no net uptake of
hydrogen was detected; PHB is not formed under
nitrogen.
When washed suspensions of these two bacilli
were shaken under air or under nitrogen, in the
absence of an exogenous carbon and energy
source, stored PHB was metabolized. The
anaerobic degradation of reserves was slower;
e.g., in B. megaterium, 61 and 17% degradation
of PHB occurred under air and nitrogen, respec-
tively, within 8 hr. Metabolic products detected
included f-hydroxybutyrate, acetoacetate, and
acetate, although aerobically CO2 and water
were the major products and only small amounts
of acetoacetate accumulated. Some correlation
was evident between the PHB content of cells
and their endogenous respiration; e.g., cells with
PHB-total N ratios of 0.83 and 3.27 had, respec-
tively, endogenous Qo2 values of 169 and 536.
Macrae and Wilkinson (46) also claimed that
N-deficient cells with a high content of PHB are
better able to withstand death and autolysis
than those with a low PHB content. Autolysis
was estimated by the total N content which, in
4 hr, fell by 12% in PHB-poor cells compared
with 5% in PHB-rich cells; the PHB content
of the latter cells decreased to the greatest ex-
tent. The method of estimation of autolysis is
not entirely satisfactory, because it has been
shown in numerous cases that nitrogenous mate-
rials are oxidized endogenously, releasing am-
monia, and that substantial amounts of nitrog-
enous compounds may diffuse from the cell
without loss of viability. Specifically in the case
of B. cereus, Clifton and Sobek showed that am-
monia is produced endogenously under some
conditions (12), and clearly B. megaterium
utilizes an endogenous substrate other than,
although concurrently with, PHB, since the
oxygen consumption is in excess of that required
for complete combustion of PHB; this other
substrate is not polysaccharide (47). It is pos-
sible, however, that autolysis may have occurred
to a similar extent in both PHB-rich and PHB-
poor cells, but the greater amount of reserve
material in the PHB-rich cells permitted utiliza-
tion of the liberated nitrogenous material and
128 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
some limited cellular synthesis occurred. Against
this possibility may be set the authors' observa-
tion that growth did not occur when PHB-rich
cells were held in a medium lacking a carbon
and energy source; growth was measured by
total N so that in these bacilli PHB may serve
as a reserve of energy but not as a source of
carbon skeletons for synthesis.
Doudoroff and Stanier (19) have made some
interesting observations concerning the role of
PHB in oxidative assimilation by Pseudomonas
saccharophila and in photoassimilation by
R. rubrum. They found with most substrates that
a major portion of the assimilated carbon (60 to
90%) initially accumulates within the cells as
PHB; when the exogenous substrate is removed,
a rapid intracellular degradation of the polymer
occurs, suggesting a physiological role as a re-
serve material. When cells are subjected to
standard fractionation procedures, the chemical
properties of PHB result in its appearance in the
hot trichloroacetic acid insoluble fraction (pro-
tein fraction). This fact had led Wiame and
Doudoroff (91) earlier to conclude that C14 is
incorporated into nitrogenous materials during
oxidative assimilation.
Incubation of starved washed suspensions of
R. rubrum with C'4-acetate allowed deposition
of 70% of the assimilated C'4 into PHB, with no
significant dilution. With C'4-butyrate, some
dilution occurred, and the polymer contained
only 58% of the assimilated C14. The fate of the
polymer in the light over a period of 12 hr was
studied under a variety of conditions, e.g., in the
presence and absence of a source of organic sub-
strates but in the presence of a N source and
CO2. The absence of an exogenous organic carbon
source led to the disappearance of more than 90%
of the polymer, but much of the C14 of this
material was redistributed into other cellular
components. The authors did not study the fate
of PHB in both nitrogen and carbon starvation,
or in the dark, so its behavior under these con-
ditions is not yet known. The rate of degrada-
tion of PHB and its conversion to other cellular
components wasdecreased when an exogenous
source of butyrate was supplied. In marked con-
trast, succinate, which is also metabolized under
these conditions, was quite unable to prevent
polymer breakdown and transfer of its C skele-
tons to other cell constituents; these processes
occurred to about the same extent as in the
absence of an exogenous carbon source. The
reason for this became apparent when it was
shown that succinate is photoassimilated princi-
pally to a glycogen-like polysaccharide, and the
dry weight of the cells increased by 40%.
Similar studies with P. saccharophila revealed
that washed suspensions of glucose-grown cells
incorporated 66% of the carbon assimilated from
U-C14-glucose into PHB, and without appre-
ciable dilution. An even greater amount (about
80%) of the assimilated carbon from acetate or
butyrate appeared in the polymer. Although ex-
perimental details were not given, the authors
claimed that tracer experiments indicated the
role of PHB as a substrate for endogenous metab-
olism in the absence of an exogenous carbon
source; its metabolism was reported to be much
slower than in R. rubrum, and transfer of polymer
carbon to other cell constituents could not be
demonstrated.
The use of PHB as an endogenous store of
carbon skeletons for synthesis in R. rubrum was
studied further by Stanier et al. (78), who showed
that for conversion of stored PHB to other cell
materials CO2 is essential. This was demon-
strated with cells which had assimilated acetate
and were then incubated in the presence of (i)
NH4Cl and He, (ii) NH4Cl and He-CO2 mixture,
and (iii) He-CO2 mixture in the absence of a
nitrogen source. After 16 hr, very small changes
in total dry weight had occurred and, in the
absence of C02, the PHB content of the cells
fell slightly but with no increase in the carbo-
hydrate or nitrogen content. In the presence of
CO2 without a nitrogen source, the PHB content
fell by about 50% and the carbohydrate content
showed a corresponding gain, but there was no
change in nitrogen content. WNhen both nitrogen
and CO2 were furnished, the PHB disappeared
almost completely with concomitant increases in
both carbohydrate and nitrogen content, includ-
ing protein. None of the experiments was de-
signed to test the possibility that PHB may
serve as an energy store, as it does, perhaps, in
B. megaterium and presumably also in P. sac-
charophila. It is considered that PHB serves as a
store of carbon and reducing power for further
CO2 assimilation, which is essential for PHB
utilization. The anaerobic utilization of PHB in
the absence of CO2 and a nitrogen source would
appear to be limited, but neither its formation
nor degradation aerobically in the dark has been
VO0L.- 28, 1964 129
DAWES AND RIBBONS
studied, and here one might expect PHB to
serve as a source of energy.
The presence of PHB as an endogenous reserve
in Hydrogenomonas is documented by the work
of Schlegel, Gottschalk, and von Bartha (73).
When chemolithotrophic growth, in an atmos-
phere of H2-02-CO2 (60:30:10), is limited by
nitrogen exhaustion (NH4Cl) in the medium, cells
in the stationary phase continue to increase in size
although no division occurs. The increase in dry
weight is accounted for entirely by PHB forma-
tion. Washed suspensions synthesize PHB from
CO2 by oxidation of hydrogen, and the stoichi-
ometry corresponds to:
25H2 + 802 + 4CO2 -- (C4H602) + 22H20
The rate of endogenous respiration of PHB-
poor cells (about 10% of the dry weight) was
considerably less than that of PHB-rich cells
(about 50% of the dry weight). It was little
affected by the addition of a nitrogen source
(NH4Cl) which had a marked stimulatory effect
on the respiration of the latter cells. Determina-
tions of cellular carbon and total nitrogen during
endogenous respiration, and during hydrogen
oxidation in the absence of C02, were carried
out with PHB-rich cells, both with and without
a nitrogen source. In air, only about 8% of the
PHB was consumed in 12 hr by endogenous
metabolism, but with added NH4Cl the total
cellular nitrogen increased significantly and the
PHB decreased by some 73%. Under an at-
mosphere of C02-free H2-02, the increase in cell
nitrogen was much greater, although not much
more PHB was utilized (76 %). These results
suggest that in Hydrogenomonas stored PHB may
serve as a carbon and energy source and can
support protein synthesis in the presence of a
suitable source of nitrogen. When hydrogen is
provided as an additional energy source, the
efficiency of protein synthesis is increased.
Conditions for the accumulation of PHB in the
halophile, Micrococcus halodenitrificans, were
determined by Sierra and Gibbons (75). These
authors further studied the role of PHB and its
relationship to endogenous respiration and sur-
vival of the organism (76). The RQ of the en-
dogenous respiration of M. halodenitrificans was
0.87 i 0.05. (That required for complete com-
bustion of PHB is 0.88.) Aeration of washed
suspensions at 25 C reduced the PHB content
slowly (from 55 to 29%G in 127 hr); under these
conditions, some lysis of cells occurred and the
endogenous Qo2 remained at 40 throughout this
period. Some product of metabolism or lysis
was inhibiting further endogenous respiration,
since the inhibition could be relieved by washing
the cells. Changes in the cell constituents of
PHB-poor cells (containing 10% PHB) showed
that 3 hr of starvation produced no changes in
total N, either soluble material or carbohydrate,
but almost half the PHB had been consumed.
The endogenous Qo2 value decreased with poly-
mer depletion. A similar but extended study
was made with PHB-rich cells; after 96 hr of
starvation had elapsed, the PHB content had
diminished from 50 to 10% of the dry weight, and
the endogenous Qo2 remained constant. Further
starvation rapidly reduced the endogenous Qo2.
From these and other data, it seems clear that
the rate of PHB oxidation is slower during
starvation experiments than in the respirometer:
if, as appears to be the case, PHB is the sole
substrate for respiration, cells containing 50%
of their dry weight could respire for only 15 hr
with a Qo2 of 40.
Biosynthesis of PHB. In view of the unique
occurrence of PHB as a storage polymer (or
metabolic shunt product) in bacteria, it is some-
what surprising that, at the time of writing,
there is very little information regarding the
metabolic routes and enzymes concerned with
its biosynthesis. The only paper of significance
is that of Merrick and Doudoroff (56). They
demonstrated that polymer particles (obtained
by differential centrifugation of lysozyme-treated
bacteria) of B. megaterium KM were able to
incorporate C'4-f-hydroxybutyryl-coenzyme A
(CoA) into PHB. This was independent of the
presence of the soluble fraction of the cell. Ap-
proximately 40% of the total radioactivity be-
came incorporated into the polymer, which cor-
responded to the amount of thioester decomposed.
A mixture of labeled g-hydroxybutyrate and un-
labeled CoA did not allow incorporation of C14
into the polymer.
Similar experiments by these authors were
extended to the particulate fraction of R. rubrum
which contained all the activity for the incor-
poration of f-hydroxybutyryl-CoA into PHB.
However, these particles also contained a very
active depolymerase which could degrade the
PHB. With crude extracts, supplemented with
CoA, adenosine triphosphate (ATP) and reduced
130 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
nicotinamide dinucleotides, a small incorporation
of C'4-acetate into polymer occurred. Activa-
tion of f3-hydroxybutyrate could not be demon-
strated.
Although activation of ,3-hydroxybutyrate
could not be demonstrated in extracts of R. ru-
brum, whole cells of Hydrogenomonas incorporate
l3-hydroxybutyrate into PHB (73). Crotonic
acid can also be used as a substrate; in eithercase, the presence of hydrogen is not mandatory.
Presumably, the oxidation of a portion of these
substrates provides the energy necessary for
polymerization. The utilization of crotonic acid
is particularly interesting since there may exist a
2n ATP y 2n CH3CO2H
2n ADP 2n CH3CO-SCoA
Acetyl GoA
V HSCoA
n CH3COCH2CO- SCoA
Acetoacetyl CoA
_______- - - V2n[H]
CH3CHOHCHC0,H-I nCH3CHOHCH2CO' SCoA
4-Hydroxybutyrate 4-Hydroxybutyryl GoA
,, .4HSCoA
CH3CH-CHCO2H----CHCH-CH-COSCIoA1
Crotonate Crotonyl CoA (CH3- H- CHaCO-)n
I 9
Hydrogenomonas Poly-A-hydroxybutyrate
FIG. 1. Biosynthesis of poly-f3-hydroxybutyrate.
possible pathway of 13-hydroxybutyrate synthesis
omitting acetoacetate.
CH3-CH = CHC02H + H20
-* CH3 CHOHCH2 CO2H
Reactions leading to PHB are schematically
shown in Fig. 1.
Catabolism of PHB. As with the biosynthesis
of PHB, comparatively little is known of its
degradation. There are, however, indications
that the initial stages of depolymerization are
extremely complex and appear to be bound up
with the structural integrity of the polymer
particles (56, 94). Thus, Merrick and Doudoroff
(56) demonstrated that extracts of R. rubrum
contain enzymes that degrade (i) native PHB
from B. megaterium or (ii) boiled PHB particles
from R. rubrum, but do not degrade the purified
polymer.
Sierra and Gibbons (76) demonstrated PHB
esterase (depolymerase) activity in Ml. halode-
nitrificans by the anaerobic release of CO2 from
bicarbonate buffer. A stoichiometric release of
CO2 by l3-hydroxybutyrate was recorded; this
depolymerization is inhibited by the esterase
inhibitor, diethyl-p-nitrophenyl phosphate (para-
oxon). Further, the rate of release of f3-hydroxy-
butyrate was identical with the rate at which
oxygen is consumed for the complete combustion
of PHB. For example, 0.76 umole of ,3-hydroxy-
butyrate was liberated per hr by 2 mg of cells
containing 50% of their dry weight as PHB;
this requires an oxygen consumption of 3.4
Mmoles per hr for complete combustion. This is a
figure realized by the recorded Qo2 values of 39
[(39 X 2)/22.4 Mmoles of 02 per 2 mg of cells per
Poly - hydroxybutyrate
I N PHB esterase(depolymerase)
D- (-)-A - I ydroxybutyrate
r NAD
4,- I4ydroxybutyrate
NADH2 dehydrogenase
Acetoacetate
ATP, HSCoA, Mg2+
Acetyl GoA
Oxaloacetate - 5 Citrate
Tricarboxylic,
Acid
Cycle
FIG. 2. Catabolism of poly-f3-hydroxybutyrate.
hr = 3.48]. On this basis, the depolymerization
appears to be rate-limiting. Sierra and Gibbons
later showed that depolymerase activity is
markedly dependent upon Nat and Lit ions (76,
76a). Cells washed and resuspended in KCl or
water do not oxidize their PHB reserves unless
Na+ or Li+ is added.
Enzymes involved in the degradation of /3-hy-
droxybutyrate were also demonstrated by Sierra
and Gibbons (76). DL-f3-Hydroxybutyrate is
oxidized by crude extracts to acetoacetate by
a nicotinamide adenine dinucleotide (NAD)-spe-
cific enzyme. Only the D(-)-f-hydroxybutyrate
is utilized, and the acetoacetate is not further
metabolized unless ATP, Mg2, CoA, and oxalo-
VOL. 28, 1964 131
DAWES AND RIBBONS
acetate are added to the extracts. These data
suggest a pathway for catabolism of PHB as
shown in Fig. 2. f3-Hydroxybutyrate and aceto-
acetate were also detected as products of PHB
degradation in B. megaterium when cells were
starved under N2. These acids accounted for over
80% of the polymer depleted under these condi-
tions. Some properties of a D-(-)-O-hydroxy-
butyrate dehydrogenase from B. megaterium have
been described (24).
GLUCOSE - N H4* salt
reaction is optimal over a pH range of 6.8 to 8.5.
The dehydrogenase is insensitive to sulfhydryl
group reagents; ethylenediaminetetraacetic acid
(EDTA) inhibition may be reversed by Mg2 if
incubation is not prolonged.
The specific activity of the D-(-)-3-hydroxy-
butyrate dehydrogenase is dependent upon the
cultural conditions at the time of harvest; low
specific activities are observed during periods of
PHB assimilation, but specific activities greater
GLUCOSE_-TQYPTONE TRYPTONE
0-35
0-3 151
>1~~~~~~~~~~~~~~~~~~~~~~~~~0
0O25L
E ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~10
F 3. Cdarbohydofcardtt
015
~~~~~~~~~~~~~~~~8
~0.1N/35
~0
0 I010S I10 18 6 2
Time (/77/n)
FIG. 3. Comparison of carbohydrate utilization and ammonia production by endogenously respiring cells
of Escherichia coli harvested from stationary-phase glucose-ammonium salts, glucose-Tryptone, and
Tryptone cultures (Ribbons and Dawes, 65). Reproduced by kind permission of The New York Academy of
Sciences.
Shuster and Doudoroff (74) found the D(-)3-
hydroxybutyrate dehydrogenase from R. rubrum
to be cold-sensitive. A 250-fold purification of the
enzyme was obtained in 30% yield, and this was
unstable in dilute solutions or inactivated by
freezing. The enzyme is freely reversible, and the
reaction products are acetoacetate and reduced
NAD (NADH2). Although NAD phosphate
(NADP) would not substitute for NAD, a-oxo-
valerate showed 6% of the activity obtained with
acetoacetate. Furthermore, a-oxovalerate compet-
itively inhibited acetoacetate reduction, because
of the lower affinity of acetoacetate. The rate of
by a factor of 2 are observed with older cells
depleted of polymer. Succinate-grown cells con-
tain no PHB, and only low specific activities of
the enzyme are found.
Glycogenlike Reserves
The majority of microorganisms synthesize
polysaccharides of one form or another which
may be of intracellular or extracellular origin.
Only the former type come within the scope of
the present survey since, by definition, extra-
cellular polysaccharides [reviewed by Wilkinson
in 1958 (92)] cannot serve as endogenous sub-
132 BACTERIOL. R.EV.
EN21ENDOGENOUS METABOLISM OF BACTERIA
strates. It is frequently possible to differentiate
intracellular polysaccharide into "reserve" and
"structural" carbohydrate, the criterion being
whether or not the material is utilized under con-
ditions of starvation. The term "structural" in
this context may be incorrect in many instances,
since it does not follow ipso facto that material
not metabolized is necessarily part of the archi-
tecture of the bacterial cell. The role of reserve
carbohydrates in yeast was also reviewed recently
(15).
We have investigated the role of glycogen as
an endogenous substrate in Escherichia coli and
have obtained considerable evidence for its rapid
utilization during periods of complete starvation
under either aerobic or anaerobic conditions (16,
17, 65). The possession of glycogen by the cells
prevents a net degradation of nitrogenous ma-
TrABLE 2. Oxygen consumption and carbohydrate
utilization by endogenously respiring
Escherichia coli
02 re- 02 re-
Time 02 quired for Difference quired for RQconsumed glycogen ribose corn-
combustion bustion
min jmoles jimoles Mmoles Pmoles
60 11.38 11.01 0.37 0.73 1.01
120 18.89 16.01 2.88 1.85 1.03
184 23.2 17.48 5.72 2.03 1.01
270 26.6 18.47 8.13 2.87 1.01
terials with liberation of ammonia; this was
demonstrated with cells harvested from media
allowing massive, moderate, and no deposition
of glycogen. Subsequent starvation of washed
suspensions of these cells showed that ammonia
is released only after the glycogen has been oxi-
dized, and in the case of Tryptone-grown cells,
which do not contain glycogen, oxidation of
nitrogenous materials commences immediately
upon starvation (Fig. 3). The regulatory mecha-
nisms of this phenomenon have not yet been
studied. Other data indicate that, during oxida-
tion of endogenous glycogen, very little else is
oxidized, since the consumption of oxygen and
evolution of CO2 correspond initially to that
required for the complete combustion of glycogen
to CO2 and water (Table 2). Longer periods of
starvation reveal that oxygen consumption is in
excess of glycogendepletion, and a part of this can
be accounted for by oxidation of ribose, which is
produced in the degradation of the RNA that
occurs under these circumstances.
The exact role of the glycogen under these
starvation conditions is difficult to appreciate,
since depletion of most of it is complete within
2 to 3 hr. If the possession of glycogen by E. coli
favors survival by providing a store of energy,
then one might expect a slower rate of utilization
of this storage product. However, the exact con-
ditions of starvation will influence considerably
the fate of the polysaccharide, and those used
in our experiments may favor an uncoupling of
oxidative phosphorylation or other rate-limiting
process. Even under a nitrogen atmosphere, part
of the glycogen is rapidly degraded, with the evo-
lution of hydrogen and carbon dioxide. It may
be that in E. coli the glycogen functions as a re-
serve of carbon skeletons and not of energy. Evi-
dence in support of this concept is the transfer of
glycogen carbon atoms to other cell constituents
(36).
The deposition and fate of glycogen in Aero-
bacter aerogenes was studied by Strange, Dark,
and Ness (81). Cells grown on Tryptone-glucose
media contain 15 to 20%7, carbohydrate, and most
of this is glycogen. It is utilized after approxi-
mately 25 hr of incubation of the washed suspen-
sions in buffer. Ammonia is released from glyco-
gen-containing cells, but at a lower rate than
from cells that have not stored glycogen. The
complete suppression of ammonia release by
glycogen as recorded with E. coli (65) was not
observed, although the glycogen is oxidized much
less quickly in A. aerogenes. Ultraviolet-absorb-
ing materials are also released from glycogen-
containing cells of A. aerogenes much more slowly
than from cells harvested from tryptic meat broth
or defined media (carbon-limiting).
Glycogenlike polysaccharides have been de-
scribed as reserves in a variety of bacterial
species; some examples are given in Table 3. The
amount stored was reported to be as high as 41
to 75% of the dry weight in Arthrobacter species
by Mulder et al. (58). When washed suspensions
of exponential-phase cells of Arthrobacter are
starved for 4 days at 30 C, only 40% of their
carbohydrate is utilized. At this stage, the oxygen
consumption of the suspensions is almost zero.
Thus, not all of the original carbohydrate is a
substrate for endogenous respiration.
R. rubrum, it may be recalled, is an example of
a microorganism that is able to store more than
V o ,. 28, 1964 133
DAWES AND RIBBONS
one polymer as a reserve material, and the poly-
mer that is deposited within the cells is deter-
mined solely by the chemical nature of the carbon
substrate supplied. Thus, acetate and butyrate
are substrates for PHB storage, whereas carbon
dioxide, succinate, propionate, and malate are
photoassimilated principally to a glycogenlike
polysaccharide (78).
The photoassimilation of specifically labeled
C'4-succinate by starved cells of R. rubrum was
studied in detail by Stanier et al. (78). The
polysaccharide was the most strongly labeled
fraction when either 1-C14_ or 2-C'4-succinate was
photometabolized; 75% of the assimilated suc-
cinate flowed into the polysaccharide and only
14% into PHB. Carboxyl-labeled succinate
labels the hexose residues at C-3 and C4, and
TABLE 3. Occurrence of glycogen in bacteria*
Species Reference
Escherichia coli .......... 35, 36
Aerobacter aerogenes .. 81
Rhodospirillum rubrum ... 78
Arthrobacter sp...................... 58
Agrobacterium tumefaciens .. 48
Bacillus cereus...................... 58
B. megaterium...................... 3
Mycobacterium phlei ............. ... 25
M. tuberculosis...................... 11
* This is not a complete list of the bacterial
species that store glycogen.
the methylene carbon atoms of succinate enter
C-1, C-2, C-5, and C-6 of the glucose residues.
The specific activities of the incorporated ma-
terial showed that the carboxyl-labeled succinate
is diluted by a factor greater than two, whereas
the methylene carbon atoms are diluted only
slightly. The main mechanism of hexose syn-
thesis, therefore, seems to involve decarboxyla-
tion of the succinate chain (probably as oxalo-
acetate), the product of which is channelled by
reversal of the reactions of glycolysis to hexose
phosphate.
The chemical nature of the carbon nutrient
determines the storage product formed; those
compounds that are converted to acetate without
intermediate formation of pyruvate (or phos-
phoenolpyruvate) yield PHB, and those yielding
the 3-carbon compound produce glycogen. Dur-
ing photometabolism in R. rubrum, glycogen
serves as a source of carbon and reducing power
for further CO2 assimilation; it may also provide
some energy, although this has not been tested.
It appears to represent a "hot-house" shunt
product (as does PHB in R. rubrum) as suggested
by Foster (22), since massive deposits are formed
rapidly by assimilation of exogenous carbon
sources at rates greater than overall cellular syn-
thesis. The fate or synthesis of the glycogen has
not been studied during aerobic metabolism of
this organism; here, as with PHB, the glycogen
might be expected to serve additionally as an
energy source.
Hot water or hot 75% ethanol extracts a poly-
glucose compound of low molecular weight from.
glucose-peptone grown Sarcina lutea (5, 9, 65).
We have shown that this polyglucose is only
synthesized during growth on peptone media
supplemented with glucose, and that it can serve
as a substrate for endogenous respiration. The oxi-
dation of this carbohydrate occurs during the de-
pletion of the amino acid pool; i.e., the provision
of a readily metabolizable carbohydrate as an
endogenous reserve does not spare the nitrogen
reserves, since free ammonia is released during
the starvation period. Further, the oxygen con-
sumption is in excess of that required by the
polyglucose oxidized.
The metabolic pathways of glycogen biosyn-
thesis and degradation in bacteria have received
very little attention. Rogers (66) suggested that
bacterial enzymes degrading glycogen are un-
known, although many systems that decompose
starch, amylose, and amylopectin are described.
Glycogen metabolism is, however, well docu-
mented in animals and plants. [For reviews see
Stetten and Stetten (79) and Whelan (90).]
Current ideas of glycogen metabolism suggest
that biosynthesis and dissimilation occur by
separate routes. The uridine diphosphoglucose
(UDPG) pathway is involved in synthesis and
the phosphorylase pathway in degradation. The
initial reactions of glycogen breakdown were
delineated by Parnas and co-workers as early as
1935, when they showed that inorganic phos-
phate is consumed and a hexose monophosphate,
later identified as glucose 1-phosphate, is accumu-
lated. The reaction catalyzed by glycogen phos-
phorylase is:
Pi + glucosyl-(al1,4') primer
glucose 1-phosphate + primer
In mammalian systems, complex interrelation-
134 BACTERIOL. REV.-
ENDOGENOUS METABOLISM OF BACTERIA
ships exist between inactive and active forms of
phosphorylase, and the enzymes obtained from
different tissues of the same species are not iden-
tical (79). The bacterial phosphorylases are, how-
ever, not well studied.
The UDPG pathway of glycogen synthesis has
been described in bacteria by Madsen (48, 49),
and in yeast by Algranati and Cabib (1). Glyco-
gen acts as a primer with the glycogen synthetase
enzymes of Agrobacterium tumefaciens and yeast,
but, unlike the mammalian enzymes, glucose
6-phosphate does not stimulate the reaction:
UDPG + polysaccharide primer
uridinediphosphate (UDP)
+ glucosyl (1,4') primer
A cyclic scheme of glycogen degradation and
synthesis has been proposed for mammalian
UDP GLYCOGEN
UDPG glycogen VI
synthetase
P UDPG Phosphorylase
PP
UDPG
p \rophosphorylaseUTP
Glucose-1- phosphate
1 Phosphoglucomutase
GLUCOSE E.AT . Glucose 6-phosphatelRexoki~nase
FIG. 4. Biosynthesis and degradation of glycogen.
systems (Fig. 4). A similar system probably oper-
ates in bacteria, since the necessary enzymes have
been demonstrated and partially separated in A.
tumefaciens (48), and UDPG has been shown to
be a competitive inhibitor of phosphorylase in the
same organism, suggesting that the concentration
of UDPG may regulate glycogen synthesis not
only directly, but also by inhibiting the degrada-
tive enzymes (49).
Madsen (50) has provided further evidence to
support the postulate that the control of glyco-
gen metabolism is effected by UDPG. The UDPG
pathway to glycogen is essentially irreversible,
and the equilibrium of the phosphorylase reac-
tion is slightly in favor of glycogen synthesis,
yet the concentration of glycogen in A. tume-
faciens varies considerably. Madsen analyzed A.
tumefaciens during its growth in batch culture for
glycogen and UDPG content. Both increase
initially during a short lag phase and then de-
crease at the beginning of exponential growth.
In the stationary phase (growth limited by nitro-
gen), the concentrations of both compounds rise
again, the UDPG slightly before the glycogen. A
linear relationship between glycogen concentra-
tion and UDPG concentration was demonstrated.
Replenishment of the nitrogen supply in the
stationary phase in another experiment caused
resumption of growth, depletion of UDPG, and
cessation of glycogen synthesis. During aeration
of washed suspensions of A. tumefaciens in buf-
fered salt solution, the glycogen was utilized.
The UDPG concentration during this period re-
mained low and constant.
Glycogen is a highly branched polysaccharide,
and little or no reference has been made to the
synthesis of (al ,6') links; the UDPG synthetase
will only synthesize (al,4') bonds although a
branched primer is required. A branching en-
zyme similar to Q-enzyme has been described in
yeast which will synthesize glycogen from amylose
by transglucosylation (al ,4' to al,6').
Amino Acid Pools
The first indication that the free amino acid
pool could serve as a source of substrates for
endogenous respiration was provided by Dawes
and Holms (14). Aeration of washed suspensions
of stationary-phase, peptone-grown S. lutea re-
duced the endogenous Qo2 values to negligible
levels, during which time the free amino acid pool
was depleted to one-half its original level and
ammonia was released into the supernatant fluid.
Hydrolysis and analysis of the hot water-soluble
pool also showed that some peptide material was
being used as substrates of endogenous respira-
tion. The total oxygen consumption during the
period of endogenous respiration corresponded to
7.5 and 3.4 jsmoles of oxygen per Emole of utilized
amino acid in the unhydrolyzed and hydrolyzed
pools, respectively. The latter value is a reasona-
ble average figure for the oxidation of a mix-
ture of the amino acids that are utilized during
the starvation.
Glycine, threonine, leucine, tryptophan, and
a-aminobutyric acid were completely utilized,
and much of the serine, glutamate, and alanine of
the pool was oxidized. Very little loss of amino
acids to the suspending media occurred (about
2% of the initial concentration of the hydrolyzed
pool). Glutamate plays a most important role in
the endogenous respiration of S. lutea; it accounts
for 20% of the total pool amino acids in freshly
VOL. 28, 1964 135
DAWES AND RIBBONS
harvested peptone-grown cells, and this declines
to 1% after a 5-hr aeration period.
Although glucose-peptone grown S. lutea also
stores a polyglucose compound as a reserve mate-
rial, the free amino acid pool is depleted just as
rapidly during starvation of these cells (9, 65).
The utilization of free amino acid pools as en-
dogenous substrates has since been shown to occur
in Nocardia rugosa (2) and Staphylococcus aureus
(64). During starvation of washed mycelial sus-
pensions of N. rugosa, both the endogenous Qo,
and Qo, (glucose) fall by approximately the same
value. There is a loss of weight from the mycelia
that can be accounted for almost completely by
loss of protein; a little acid-soluble carbohydrate,
mainly hexose, is also utilized. There was no sig-
nificant utilization of lipid during starvation for
16 hr. The pH value changed from 7.5 to 8.2
during the incubation, and free ammonia was
formed at the expense of the mycelial protein.
Oxo-acids did not accumulate but were oxidized.
Chromatography of the free amino acid pools
showed that fresh mycelia contained large
amounts of glutamate, alanine, aspartate, leu-
cine, and valine, whereas only smaller amounts
of leucine, alanine, and glutamate remained after
starvation. The RNA and DNA contents of the
cell are not utilized endogenously. It was con-
cluded that the main substrates of endogenous
respiration are the amino acids that are present
in the pool, and also obtained from the hydrolysis
of cell protein; an acid-soluble carbohydrate is
also utilized. The presence of an exogenous source
of glucose inhibits the endogenous consumption
of protein in N. rugosa.
Ramsey (64) showed that washed suspensions
of S. aureus respire endogenously with an RQ of
0.83 to 0.86. The carbohydrate content of the
cells remained constant (at the very low value of
1.18% of dry weight) during starvation, and
ammonia was released into the supernatant fluid.
The 02-NH3 ratio was 6.2, and glutamate was
utilized from the free amino acid pool. This indi-
cated that substances other than glutamate were
being utilized (02-NH3 ratio for glutamate is
4.5). The endogenous respiration of incompletely
C'4-labeled cells confirmed this view, since apart
from the utilization of glutamate in the pool
there was also some loss of C14 from the hot tri-
chloroacetic acid-insoluble fraction of the cells;
further, the oxygen consumption was much in
excess of that required for oxidation of glu-
tamate alone (glutamate can account for only
10% of the 02 consumed). More completely
labeled cells were obtained by growth on U-C14-
glucose and C'4 algal hydrolysate, and the change
in cell components during respiration was fol-
lowed. Only about one-third of the radioactivity
lost from the cells is recovered as C'402. The rest
can be traced to the suspending buffer, and rather
more than one-third to deposition in the hot
trichloroacetic acid fraction. The bulk of the
radioactivity was lost from the hot trichloroacetic
acid-insoluble fraction. Apart from the utilization
of glutamate from the free amino acid pool, evi-
dence was presented to show that aspartate was
a probable substrate and, to a lesser extent,
alanine. Cells, allowed to assimilate C'4-glycine
into the pool, washed, and subsequently starved,
liberated C1402 only slowly.
Comparisons of the endogenous metabolism of
coagulase-positive (+) and -negative (-) S.
aureus have revealed marked differences (D.
Ivler, personal communication). During starvation
of organisms grown on Brain Heart Infusion, the
RQ of + cells showed little change (from 1.08
to 0.95), whereas that of - cells fell from 1.10 to
approximately 0.5 in 60 min. The Qo2 of + cells
was higher than that of - cells, but both values
decreased rapidly with starvation. High rates of
endogenous respiration of + cells suppressed the
rate of oxidation of added glucose, but no such
effect was apparent with - cells. Measurement
of 02-NH3 ratios during starvation of washed
suspensions showed that values fell from 4.7 to
3.0 with + cells and from 13.4 to 10 with - cells.
Of considerable note was the fact that while the
free amino acid pool content of both types of
cell diminished, the bulk of the loss could be
accounted for as free amino acids in the super-
natant fluid (as much as 90% recovery with -
cells); nonetheless, ammonia appeared in thesupernatant at a greater rate and to a greater
extent with + cells, indicating the net degrada-
tion of nitrogenous materials. The total carbo-
hydrate content of both + and - cells did not
alter during starvation.
RNA Metabolism
Mlore typical storage compounds such as
glycogen or PHB are characterized by their
deposition during conditions of carbon source
excess or nitrogen limitation, and by their de-
pletion to almost negligible amounts during
136 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
starvation. However, as Herbert (34) empha-
sized, the amounts of such basal materials of the
bacterial cell as RNA, DNA, and protein are
subject to wide variation, and this can be con-
trolled by environmental conditions.
Strange, Dark, and Ness (81) showed that
RNA is metabolized endogenously during starva-
tion of washed stationary-phase suspensions of
A. aerogenes, and net degradation occurs. The
extent of RNA catabolism varied, and this
depended on the source of the cells; 40% of the
RNA of cells harvested from defined media was
utilized in 70 hr, during which time about 70%
of the population remained viable. [See also E.
coli (7)]. Cells harvested from glucose-Tryptone
media contain much less RNA (about 11% of
the dry weight), and little is utilized. These cells
contain glycogen that is almost completely
respired within 25 hr. Tryptic meat broth grown
A. aerogenes, on the other hand, catabolize nearly
half their RNA, from about 13% to 7% of the
dry weight, within about 50 hr. The products of
RNA metabolism that have been detected in the
suspending fluid include ammonia, inorganic
phosphate, and the free bases hypoxanthine,
uracil, and guanine (62, 81, 83), and small
amounts of adenine (83); hypoxanthine was the
major component of the bases in the supernatant
(83). Nucleotides and nucleosides did not ac-
cumulate to any significant extent, and most of
the pentose was apparently oxidized. The ultra-
violet-absorbing materials were readily released
into the supernatants, and acid-soluble inter-
mediates did not accumulate within the cells (83).
The amount of ultraviolet-absorbing material
released corresponded well with the amount of
RNA lost from the cell, and this loss occurred
almost entirely from the RII sedimentation
fraction (83), as had been demonstrated in the
case of loss of RNA from E. coli ribosomes (85).
RII is the cell fraction sedimented at 78,000 X g
for 7.5 hr.
The endogenous utilization of RNA is not
peculiar to A. aerogenes, but appears to be very
widespread, and has been demonstrated in E.
coli (17, 18), S. lutea (9), and P. aeruginosa (27).
The utilization of the ribose portion of the
RNA by endogenously respiring E. coli was
briefly mentioned in the section on glycogenlike
reserves, and in Table 2. It is not yet clear
whether the purine and pyrimidine bases are
completely oxidized, although this is unlikely as
ultraviolet-absorbing compounds accumulate in
the supernatants. The degradation of RNA in
E. coli was studied by Wade (85), who concluded
that two pathways exist. The M route (Mg2+-
dependent) results in the formation of nucleo-
side 5'-phosphates characteristic of phosphodi-
esterases. The rate of formation of these nucleo-
tides is further stimulated by inorganic phos-
phate, suggesting that polynucleotide phos-
phorylase was also depolymerizing RNA (86).
Autodegradation of ribosomes in the presence of
inorganic P32-orthophosphate gave only labeled
nucleoside diphosphate. The nucleoside mono-
phosphates appeared to be formed by an inde-
pendent route (86). The second route, the V
route, results in the degradation of RNA into
nucleoside 2', 3'-cyclic phosphates in the presence
of sufficient EDTA to remove the M\g2+. The
cyclic phosphates are further hydrolyzed to
nucleoside 3'-phosphates. The V route then
employs ribonuclease-type enzymes. The enzymes
of both routes are located in the ribosomal frac-
tion. The observation of Kiguchi and Uemura
(84a) that citrate and phosphate enhance the
release of RNA degradation products from yeast
cells is, perhaps, relevant. These authors believe
that magnesium is removed from the cell
membrane by chelation, since added Mg2+
countered the effect of these agents.
Starving washed suspensions of P. aeruginosa
release much ultraviolet-absorbing material into
the supernatant with an Emax at 260 mIu. Nucleo-
tides, nucleosides, and free bases were detected.
The effect of Mg2+ ions on ribosomal particles is
well known (8), and Gronlund and Campbell (27)
have used this as evidence for the utilization of
RNA as a substrate for endogenous respiration.
Oxygen consumption is depressed in the presence
of Mg2+ ions, which allow the formation of 70S
ribosomes. When P. aeruginosa was grown in the
presence of C14-uracil, the radioactivity was con-
tained primarily in the nucleic acid fraction, and
this yielded C'402 during subsequent starvation.
The C0402 was derived solely from the RNA, and
came largely from the ribosome fraction. The
enzymatic degradation of ribosomes was demon-
strated and inhibited by EDTA; phosphate
markedly increased ribosome degradation, sug-
gesting a role for polynucleotide phosphorylase.
The fate of the ribose portion of the RNA was
not determined, although ribose (free and com-
bined?) was detected in supernatant fluids.
137VOL. 28, 1964
DAWES AND RIBBONS
By calculations based on the values of C1402
released from uniformly C'4-labeled cells, 2-C04-
uracil-labeled cells, and U-C'4-proline-labeled
cells, Gronlund and Campbell deduced that the
C'402 liberated from 2-C'4-uracil-labeled and
from U-C14-proline-labeled cells is equivalent to
the total amount of C1402 liberated by uniformly
labeled cells. From this, they infer that RNA and
protein are the only endogenous substrates
oxidized. Their calculations are based, however,
on the assumption that the fate of the 2-C of
uracil is representative of all RNA carbon atoms,
since, for example, the ribose was presumably
unlabeled by this technique and its fate was not
determined. The fate of the proline C atoms is
also assumed to reflect the destiny of all the other
carbon atoms of protein.
Protein
The net utilization of protein as an endogenous
substrate was first demonstrated by Strange et
al. (81) with starving suspensions of A. aerogenes,
and Gronlund and Campbell (26) indicated that
ammonia release by endogenously respiring cells
was a general phenomenon. Thus, washed sus-
pensions of E. coli, P. aeruginosa, P. fluorescens,
Achromobacter sp., B. subtilis, and S. faecalis all
liberate ammonia during endogenous respiration
(15, 26, 87). To this list may be added A. aerog-
enes (81) [although Gronlund and Campbell (26)
did not detect ammonia production with their
strain], B. cereus (12, 47), S. lutea (14), S. aureus
(64), and N. rugosa (2). Even so, the original
work of Strange et al. is the most decisive demon-
stration of protein degradation, since they used
simple chemical analysis. Other workers have
concluded from radiochemical evidence that
protein is a substrate of endogenous respiration;
in some bacteria, it appears to be the main
substrate. With P. aeruginosa, Gronlund and
Campbell (27) labeled the cells with U-C14-proline
and demonstrated the endogenous evolution of
C1402 from the hot trichloroacetic acid-insoluble
fraction. It is interesting to note that the alcohol-
soluble protein of P. aeruginosa accounts for 20%
of the total protein (60) and yet this is not utilized
during starvation. The hot trichloroacetic acid-
insoluble residue yields less C1402 when cells are
starved in the presence of Mg2+; under these con-
ditions, there is also a slight decrease in the alco-
hol-soluble protein. Pine (60) demonstrated that
the alcohol-soluble proteins of E. coli do not dis-
appear during starvation; the solubility proper-
ties of this fraction alter, however, and cautiousinterpretations with respect to the fluctuations of
this fraction are required.
Strange et al. (83) showed that protein is lost
from all ultracentrifugal fractions during starva-
tion of A. aerogenes; i.e., ribosomal and soluble
proteins are utilized. The products of protein
catabolism appear to be ammonia and carbon
dioxide, as only traces of amino acids accumulate
in the suspending fluid.
Attention must be directed to the data ob-
tained by Strange et al. (81) for the release of am-
monia from cells grown in different media. If, on
the basis of their figures, a calculation is made to
compare the nitrogen accounted for as NH3 with
the nitrogen of the degraded protein and RNA,
considerable discrepancies are apparent. Thus
78.8, 25.2, and 30.2% nitrogen are unaccounted
for as NH3 in cells harvested from glucose-Tryp-
tone, Tryptone meat broth, and defined media,
respectively, after 25 hr of starvation. The fate of
this nitrogen has not been ascertained, and some
cellular redistribution might be envisaged.
Other Potential Substrates
Our knowledge of bacterial lipids is very
limited (34), especially concerning their role as
endogenous reserves of energy. It appears that
conventional lipids are stored by E. coli under
favorable conditions; e.g., provision of acetate
stimulates lipid deposition (13). The utilization of
lipid and phospholipid materials during starva-
tion has been discussed (15). The massive deposi-
tion of lipids by the yeasts Rhodotorula gracilis,
Lipomyces starkeyi, R. graminis, and R. glutinis
was recently described by Mulder and co-workers
(58).
DNA is generally considered to be a stable cell
constituent whose function is storage of informa-
tion, and generally no utilization of DNA occurs
during starvation. However, Strange, Wade, and
Ness (83) showed that the DNA of starving A.
aerogenes increased by 17%, and they ruled out
the possibility of cell division occurring. A DNA
increase at the expense of RNA was also demon-
strated with phosphate-deficient E. coli (37). On
the other hand, utilization of DNA after 19 hr of
starvation was noted in A. aerogenes (30).
ENERGY OF MAINTENANCE
Cellular processes, whether mechanical or
chemical, require energy for their performance,
138 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
and unless a supply of energy is readily available
these essential processes will cease and the cell
will die. The provision of energy by exogenous
nutrients is well established, e.g., the use of glu-
cose as an energy (but not carbon) source by
Streptococcus faecalis (4). Under conditions of
starvation, mobilization of endogenous sub-
strates must furnish the energy necessary to allow
the various cellular activities to continue. The
energy required for these processes of cell sur-
vival has been called the "energy of maintenance."
Since the performance of work is required for
all these activities, which include resynthesis,
osmotic regulation, and heat loss to the external
environment, presumably they will eventually
cease owing to lack of energy, and the cell will
no longer be able to maintain its status quo. To
maintain the intact living cell, structures such as
the cell wall, flagella, cell membrane, and cell
particles must be kept in good repair. It is pos-
sible that some of these structures may be dis-
pensed with for the sake of survival; indeed,
flagella have been removed from bacterial cells
without affecting viability (45), while suitably
prepared protoplasts possess metabolic activities
that are identical with those of whole cells (71).
The possession of a rigid cell wall provides dis-
tinct advantages, since protoplasts remain intact
only in solutions of high osmotic pressure. The
cell wall is probably an essential feature of cell
survival under natural conditions. Protoplasts
are able to grow (increase in size) but apparently
are unable to undergo cell division unless rever-
sion to bacillary form (in gram-negative organ-
isms only) occurs (55a).
Mandelstam (52) reported that starved sus-
pensions of E. coli break down and resynthesize
their macromolecular components (RNA and
protein) at a rate of 5% per hr. On the other hand,
growing cells appear not to exhibit any appreci-
able turnover of protein. The continued resynthe-
sis of macromolecular components during starva-
tion requires energy, which may be supplied by
the components that are undergoing transforma-
tion. This situation pinpoints a feature not gen-
erally appreciated, namely, that the maintenance
requirement is not necessarily a constant feature
independent of growth rate; i.e., at the extremes
of unrestricted growth and absence of growth,
protein turnover (and, therefore, the energy re-
quired for this process) varies from 0 to 5% per
hr. Consequently, unrestricted growth should
have a lower maintenance requirement.
Microorganisms are able to survive for con-
siderable periods during starvation and conse-
quently must maintain soluble constituents, often
against considerable concentration gradients.
The regulation of the cytoplasmic osmotic pres-
sure and pH value appear to be highly selective
processes, since considerable quantities of some
cell substances, e.g., RNA from bacteria and
yeast, and also smaller molecular moieties, such
as amino acids and bases of RNA, ribose, and in-
organic phosphate, are able to diffuse into the
suspending fluids without loss of viability (7, 17,
27, 68, 81).
For bacteria to remain motile, a source of
energy is required; tactic responses were dis-
cussed at some length by Weibull (89). In the
absence of exogenous substrates, energy must be
supplied from endogenous sources. Phototrophic
organisms present a somewhat different case in
that their source of energy is light. If light is the
sole source of energy available to green and
purple sulfur bacteria, then incubation of washed
suspensions in the dark should exclude mecha-
nisms of ATP synthesis, and it would be of in-
terest to know the survival characteristics of
these species in the dark. This situation is similar
to the survival of strict aerobes under anaero-
biosis.
Although we are principally concerned with the
concept of maintenance energy in starving cells,
it is obvious that some consideration of growing
cells should be included; additional demands for
energy may be manifest here. The actual proc-
esses of cell division may require larger amounts of
energy than the other phases of the growth cycle
of individual cells which, in the main, would be
supplied by exogenous sources. Lowered cell
yields at low growth rates may, in fact, be caused
by the diversion of energy from synthesis of cell
substances to the physical and energetic task of
maintaining a cell that spends a long period over
division. Alternatively, cells actually dividing
may dissociate energy-yielding reactions from
synthesis without affecting the rate of utilization
of energy source.
The chemical and mechanical activities of the
microbial cell, therefore, lead one to postulate
that some energy is utilized to maintain the cell in
a functional and viable condition, and that during
starvation this energy must be derived from
VOL. 28, 1964 139
DAWES AND RIBBONS
endogenous sources. We think few scientists
would now deny the energy-of-maintenance re-
quirement, although there are several widely
quoted experiments that have been cited as
evidence against such a concept, often because it
could not be detected. This is especially apparent
in the growth-yield experiments of Monod (57)
and Bauchop and Elsden (4). Microbial growth
yields are (within certain limits) directly propor-
tional to the concentration of limited nutrient.
Even when the carbon and energy source limits
the yield of cells, the relationship holds for very
low concentrations; extrapolation of the experi-
mental points indicates that no intercept occurs,i.e., at zero concentration of nutrient no growth
occurs. If some portion of the energy source were
utilized for functions other than growth, then
one should observe that the addition of very
small amounts of an energy source would not
permit growth. This then would assume that
growth is a secondary feature of energy utiliza-
tion and that energy is preferentially channelled
to maintenance purposes. The concentrations of
the energy source used in these experiments were
such that, although they limited the maximal
population attainable, they did not limit the rate
of growth. Monod also limited the rate of growth
of E. coli by limiting aeration, and although this
doubled the time taken to achieve maximal
density in one culture the cell yield was un-
changed. He concluded that since the rate of
growth did not influence the cell crop any
energy-of-maintenance values were nil.
It seems to us that many of the experiments
designed to test the use of a portion of the exoge-
nous energy source for maintenance rather than
growth are complicated by the fact that the
energy source is also a source of carbon for
growth. More definitive evidence might be ob-
tained with microorganisms which do not in-
corporate their energy source into cell substance;
there are numerous systems available for such
experiments-phototrophs, autotrophs, and the
nutritionally fastidious anaerobes that ferment
carbohydrates almost solely as a source of energy.
Furthermore, experiments designed to show that
some portion of the energy (and carbon) source
is not utilized for growth, and many do demon-
strate this, are not entirely convincing arguments
for the maintenance concept. These experiments
do not show that the energy that is diverted
from growth is utilized specifically for mainte-
nance.
It seems feasible that at slower growth rates
the cell yield might be decreased because the cell
physiology with respect to regulatory mecha-
nisms has altered in response to the changed en-
vironment, and that a portion of the energy
source is uncoupled at the enzymatic sites of
phosphor) lation; i.e., the decreased cell yield is
merely a reflection of decreased efficiency of
conversion f the energy source into high-energy
phosphate.
It is perhaps too obvious that the reactions of
energy-yielding metabolism are not always
coupled to the growth of the organism. The more
extreme cases of this are most often observed in
batch cultures that have reached a stationary
population but still consume considerable quan-
tities of substrate. This same feature is seen dur-
ing growth limitation (rate or yield) of some
nutrient other than energy source, and is most
dramatically demonstrated by washed suspen-
sions of nonproliferating cells metabolizing added
carbon or energy substrates.
Where the energy source is also the carbon
source, usage of carbon skeletons occurs without
net growth. In some cases, the carbon skeletons
are removed from the medium and stored within
the cells (assimilation) ready for utilization under
duress. However, uncoupling of assimilation has
also been observed, in that products of metabo-
lism often appear in supernatants, and presum-
ably these are lost to the cell. Further, it has been
suggested that the products of metabolism under
conditions of carbon and energy excess are shunt
products, and that assimilation into reserves
merely reflects this glut. Limitations of the rate of
microbial growth by nutrients other than the
energy source do not control the extent of oxida-
tion of the energy source; e.g., Rosenberger and
Elsden (67) showed that in tryptophan-limited
growth S. faecalis produced large amounts of
lactate.
A discussion of the theoretical principles of
continuous culture (as controlled by nutrient
limitation), and a comparison of these with results
obtained experimentally, led Herbert (33) to
postulate that A. aerogenes displays a constant
rate of endogenous metabolism during exponen-
tial growth. The curve derived experimentally
relating steady-state bacterial concentration to
dilution rate (growth rate) does not coincide with
the theoretically predicted curve. At low dilution
rates, the cell yield is less than that expected
when the carbon and energy source is the growth-
140 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
limiting nutrient. Cultures whose growth is
limited by nutrients other than the carbon and
energy source do not show this phenomenon at
low growth rates, so that the decreased cell yields
observed under these conditions are not simply a
property of the growth rate. The lowered cell
yield recorded during slow growth on a limiting
carbon and energy source was explained by sug-
gesting that, in addition to cell synthesis from
the carbon substrate, there is also a constant oxi-
dation of cell substance to C02, i.e., some turn-
over is occurring during growth. (The experimen-
tal evidence for this is considered later.) The
equation representing the exponential growth of a
bacterial culture can be modified from
dx ds
-= sx = -Y-dt dt
to
dx
du= _- k)xdt
cohere x = cell concentration; s = substrate
utilized; t = time; A = growth rate; k = a con-
stant representing the endogenous metabolism;
and Y = yield coefficient. Thus, by lowering the
dilution or growth rate, k becomes proportionally
larger in relation to A and, therefore, the cell
density falls; the cell yield is lower because pro-
portionally more cell material is oxidized relative
to the amount of limiting nutrient that is assimi-
lated. In toto, the net result is an uncoupling of
growth from oxidation of carbon substrate, since
proportionally more nutrient is oxidized than is
assimilated per cell. This idea was expanded by
Marr et al. (53), who designed experiments to
determine the value of k (these workers substitute
a for k) which they call the specific maintenance.
Mathematically, the lowered cell yield at low
dilution rates can be expressed as:
dx (u-kx Yds
-Z = (~a-k)x =-Yddt dt
Rearranging,
Y ds
x dt
Now Y. x, ds/dt, and At (or D, the dilution rate)
can all be determined experimentally, and, there-
fore, k may be evaluated.
Substitution of k into the continuous culture
equations enables plots of steady-state cell con-
centration versus dilution rate to be made that
correspond exactly with those obtained experi-
mentally (33). Evidence to interpret k as repre-
senting a constant endogenous metabolism that
occurs during growth is demonstrated by com-
paring the rates of respiration of A. aerogenes in
continuous culture at different growth rates.
Extrapolation to zero growth rate of Qo2 and
Qco2 values determined at different growth rates
in media containing limiting glycerol gave values
on the ordinate that were identical to the Qo2
(endogenous) and Qco2 (endogenous) of these
cells. It is suggested that the respiration consists
of (i) substrate oxidation that is proportional to
the growth rate and (ii) a constant rate of oxida-
tion of endogenous material, that occurs at all
growth rates. Carbon balances (details of which
were not recorded) were also claimed to indicate
that proportionally more cell carbon than sub-
strate carbon is oxidized.
The lowered cell yields at low dilution rates
(carbon and energy source limiting) have been
observed for other bacteria and for Torula utilis
(33). Marr et al. (53) noted that E. coli is unable
to maintain cell density at low dilution rates, and
they calculated the specific maintenance as 0.025
hr-1.
The carbon balances of substrate utilization at
different growth rates received attention from
Marr et al. (53) with batch cultures. U-C14-glucose
was (i) added to a batch culture, giving exponen-
tial growth; (ii) fed rapidly to a culture so that
only a small fraction would be used for mainte-
nance, giving linear growth; and (iii) fed slowly
so that a large fraction was used for maintenance,giving curvilinear growth. It had previously
been shown that the cell crops obtained
increased in the order: slowly fed cultures <
from batch cultures < from rapidly fed
cultures. The radiochemical results confirmed this
observation and also showed that more of the
glucose is oxidized to C1402 in slowly fed than in
batch, which in turn is greater than in rapidly fed
cultures. The reverse order was found for C14
assimilated by the cells. It is not entirely clear
why the batch cultures should lie between the fast
and slow feeding of energy source, but some ex-
planations may be offered. During exponential
growth in batch culture, the rate of growth is not
limited by glucose concentration and it appears
that glucose is utilized less efficiently; e.g., it is
not oxidized to CO2 immediately, possibly owing
to a limitation of oxygen concentration. Conse-
141VOL. 28, 1964
DAWES AND RIBBONS
quently, growth may cease and the stationary-
phase cells oxidize the accumulated intermediates
to CO2. Lower growth yields in batch than in
continuous or nutrient-limited cultures were also
observed by Pirt (61).
The energy required for turnover of macro-
molecules appears to account for a larger propor-
tion of the energy source that is not utilized for
growth in E. coli (53), but these workers could
not demonstrate that accumulation of methyl-
thiogalactoside was responsible for any signifi-
cant amount of energy expenditure, although
Kepes (40) noted that addition of this compound
to E. coli suspensions resulted in a doubling of the
rate of endogenous metabolism.
The earlier ideas concerning the concept of
energy of maintenance were excellently sum-
marized by Mallette (51) and McGrew and
Mallette (55). They indicated that lack of sensi-
tive techniques was among the reasons for the
difficulty of demonstration of the energy of main-
tenance. To overcome this problem, they used a
high cell density in relation to a low concentration
of carbon and energy source to study the mainte-
nance requirement of E. coli. Low concentrations
of glucose were fed to suspensions of E. coli in an
otherwise complete growth medium, and the tur-
bidity changes were recorded. When very small
additions of glucose were made, the extinction did
not alter appreciably from control cultures after
a standard time. Higher concentrations of glucose
permitted growth to occur. Thus, they demon-
strated that a threshold concentration of glucose
is required before growth can occur. Cell suspen-
sions starved with respect to the carbon and
energy source showed rapid decreases in turbidity
for 5 days, at which time only 15% of the cells
were viable, and then remained constant while
the viability continued to fall. A small addition
of glucose at 6-hr intervals, sufficient to maintain
the turbidity at a constant value, also suppressed
the rate of loss of viability; e.g., after 5 days only
20% of the cells had died. Glucose additions that
permitted a very slow growth (20% increase in
extinction over 10 days) did not prevent death of
the cells. (After 5 days, approximately 10% had
died.) Thus, it would seem that small amounts of
glucose can provide energy to maintain the cell
without allowing growth to occur. The loss of
viability that occurs during the slow growth may
be due to the interval method (6 hr) of feeding,
as pointed out by Marr et al. (53); i.e., some
breakdown of cellular material occurs before the
next glucose supplement, and this is insufficient
to allow reclamation of the lost cell materials. A
criticism which may be leveled at this type of ex-
periment is that growth may be occurring al-
though it is not revealed by turbidity measure-
ments. The number of cells dying may be such
that the turbidity undergoes no net change as
growth occurs. The influence of higher cell con-
centrations on these phenomena is not known,
and regrowth (28) may assume special impor-
tance; perhaps open systems should be considered
in experimental design.
STARVATION AND SURVIVAL
The early literature concerning the effects of
starvation upon the survival of microorganisms
was critically reviewed by Postgate and Hunter
(62). They also draw attention to the pitfalls and
difficulties that may be encountered during the
estimation of viabilities. The cleanliness of labo-
ratory ware and purity of chemicals is considered
to be critical, as trace impurities may either per-
mit growth of otherwise starving organisms (23)
or kill the cells. The growth of bacteria at the
expense of their companions has been termed
cryptic growth (70), cannibalism (28), and re-
growth (81). This growth is a function of cell
density and can considerably influence the sur-
vival behavior of starving suspensions, since a
"population turnover" may occur.
Apart from the phenomenon of regrowth, the
initial cell density of starving suspensions also
affects their death rate. Harrison (28) first showed
the relationship between cell density and death
rate of starving suspensions of A. aerogenes, and
an optimal density for survival was demon-
strated. He concluded that an interaction be-
tween individual cells favors survival, and the
work of Postgate and Hunter (62) removes any
doubt about the possibility of regrowth occurring.
The latter authors made a very thorough study
of many factors that influence the survival of
starving suspensions of A. aerogenes. They elimi-
nated ambiguity that would arise from cryptic
growth, growth on impurities, and toxicity of
suspending fluids, by a suitable choice of cell
density [20 ,ug (dry weight) of cells per ml] and
suspending fluid [saline-tris(hydroxymethyl)-
aminomethane buffer-EDTA solution]. High illu-
mination, high temperatures, high pH values, and
high potassium ion concentrations increased the
142 BACTERIOL. REV.
ENDOGENOUS METABOLISM OF BACTERIA
death rates of starving suspensions. For example,
A. aerogenes survives better at 20 C than at 30,
40, or 10 C. Anaerobiosis accelerated death, and this
was attributed to the acid conditions produced.
We also have observed that anaerobically starved
E. coli die faster than the aerobically starved sus-
pensions (18); however, the pH value during
anaerobiosis fell only from 7.2 to 6.8 with our
more strongly buffered suspensions (unpublished
data).
Various nutrients, or the previous history of
suspensions of A. aerogenes, markedly affected
the death rates. Ca2+, Mg2+, and to a lesser extent
Fe2+, when added to the saline-buffer, prolonged
the life of the cells. The slower the rate of growth
of the bacteria, the greater was their death rate
upon fasting. This applied to organisms whose
growth rate was limited by C, N, P, and S, but
with Mg2+-limited growth the reverse was true
and the cells that had most rapidly proliferated
died fastest when starved. The effect of nutrient
additives upon death rates is, however, more com-
plex and depends upon the previous history of the
cells. Thus, Postgate and Hunter (63) observed
a general phenomenon of substrate-accelerated
death, in which the addition of the growth-limit-
ing nutrient to starving suspensions increased the
death rate. Glycerol-limited cells of A. aerogenes
showed glycerol-accelerated death (metabolites
of glycerol, e.g., pyruvate, also accelerate death);
NH4+-limited cells are killed by NH4+ additions
but not by glycerol, which is slightly protective.
Phosphate-limited cells behaved similarly, as did
other carbon source-limited cells. Sulfate-limited
cells were not killed quickly by sulfate, but in-
stead showed glycerol-accelerated death. Mg2+-
limited cells were another exception, in that addi-
tion of Mg2+ actually prolonged the life of these
cells, as it does of other cells, a feature quite inde-
pendent of the nutrient that limited their growth.
Harrison and Lawrence (30) also noted that the
effect of nutrient additions to starving suspen-
sions is influenced

Outros materiais